Leishmania chagasi, as it cycles between an insect and mammalian host, must alter expression of virulence factors to survive within these diverse environments. The scientific hypothesis underlying this research proposal is that regulation of leishmania genes is controlled by stage-specific signals that either upregulate or down-regulate proteins essential for adaptation to the host environment. To address this hypothesis, we plan to study the molecular mechanism by which L. chagasi regulates the expression of the MSP gene family. The overall purpose of this Mentored Clinical Scientist Award application is to provide the applicant with the necessary didactic training program and scientific environment to embark on his independent research career in the field of molecular parasitology. The abundance of the major surface protease MSP (also called GP63) increases during growth of L. chagasi from logarithmic to stationary phase in vitro, in parallel with a developmental increase in parasite virulence. Coincident changes occur in expression ofMSP RNA. Three distinct classes of MSP genes (MSPL, MSPS, MSPC) encode MSP. We previously reported that the abundance ofMSPL RNAs is regulated by shortening of its rnRNA half-life (T1/2) during growth to stationary phase. Furthermore, culture with inhibitors of protein synthesis such as cycloheximide prolongs the MSPL RNA T1/2. Parasites transfected with reporter gene constructs showed that sequences within the MSPL 3'UTR are responsible for the cycloheximide-induced increase in MSPL RNA T1/2, possibly by interacting with a labile protein factor that destabilizes MSPL mRNA. The specific aims of this work are: Aim 1. Identify the cis-acting sequences contained within the 3'UTR of MSPL that are responsible for the cycloheximide dependant increase in mRNA abundance. Aim 2. Confirm that putative cis-acting regulatory elements are responsible for in vivo mRNA regulation. Aim 3. Isolate genes encoding protein(s) that specifically bind to the cis-acting elements identified in Aim 1. This project should provide the background to launch an independent research program focusing on cis-acting factors and trans-acting elements influencing expression of leishmania genes.